JPH0776596A - Production of peptide, fused peptide, expression vector, and recombination protein - Google Patents

Abstract

PURPOSE: To provide a peptide capable of linking with a protein without inhibiting the function of a recombinant protein and becoming an easily usable reagent and showing easily controllable detection properties.

CONSTITUTION: A peptide comprises an amino acid sequence of Trp-X-His-Pro- Gln-Phe-Y-Z (wherein X is any desired amino acid; and Y and Z are both Gly or Y is Glu and Z is Arg or Lys). A recombinant protein is obtained by using a DNA sequence coding for a fusion protein consisting of the amino acid sequence of a complete protein, of a protein mutant, i.e., a deletion or substitution mutant or part of a protein linked to this peptide sequence and, if desired, detecting the presence of the expression product by means of a conjugate of streptavidin and a label and separating the desired protein as a fusion protein by streptavidin affinity chromatography.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein fused to the peptide, streptavidin (Streptavid).
avidin) a peptide that confers binding affinity, a fusion protein containing such a peptide, and a DNA sequence encoding such a recombinant protein are expressed in a suitable host cell by a method known per se to give the recombinant protein. It relates to a method of manufacturing.

[0002]

BACKGROUND OF THE INVENTION Although systems for efficiently expressing foreign proteins in a large number of different host cells are well established, the detection and purification of recombinant gene products during genetically engineered protein production remains a problem. is there. This means that a particularly important native protein has not been previously isolated and, as a result, the biochemical properties of the protein cannot be confirmed, nor can specific antisera or monoclonal antibodies to the protein be obtained. Not applicable if not. This situation frequently occurs due to the recent development of the replication chain reaction [Bloch W.
(1991) Biochemistry 30, 27
36-2747]. This is because such development often makes it easier to obtain information about the gene encoding the protein than the purified native protein itself.

In order to be able to detect the recombinant gene product when a specific immunoreagent for the protein cannot be used, a short peptide tip fused to the recombinant protein (peptide tag = Peptid-tag).
s) was used. If such a fusion peptide sequence is attached at the amino- or carboxy terminus of the protein sequence, the peptide sequence will be recognized by the specific antibody. An example of such a peptide tip is a Myc-tag (Myc-tag) [Munro and Pelham (1
986) Cell 46, 291-300; Ward.
Et al. (1989) Nature 341, 544-54.
6], Flag-Peptid
[Hopp et al., (1988) Bio / Technol.
Ogy 6, 1204-1210], KT ³ -epito-
Peptide (KT ³ -Epitop-Peptid)
[Martin et al. (1990) Cell 63, 8
43-849; Martin et al. (1992) Sci.
255, 192-194], α-tubulin epitope peptide (α-Tubulinepito).
pPeptid [Skinner et al. (1991)
J. Biol. Chem. 266, 14163-141
66] and T7 gene 10-protein-peptide-
Tag (T7 Gen 10-Protein-Pept
id-Tag) [Lutz-Freyermuth, etc.
(1990) Proc. Natl. Acad. Sc
i. USA 87, 6393-6397], and these peptides are used for the detection of recombinant gene products.
In some cases, it was also used effectively for its purification. Furthermore, it was also confirmed in most of the above examples that these short peptide tips (usually 3-12 amino acids in length) do not interfere with the biological function of the protein and therefore do not necessarily have to be separated after expression. Was done.

Although these fusion peptides are particularly suitable for the detection of proteins and are therefore important auxiliary agents for the optimization of expression yields and purification protocols, the affinity of the peptides for their purification step sequence The scope of application of is limited. The reason for this is that the advantageous properties of the previously described peptides are based on the strong binding of these peptides to antibodies. Therefore,
When a protein is bound via the peptide tip to an affinity column with immobilized antibody, it is necessary to elute the protein again from the same column under fairly extreme and therefore less protective conditions (ie non-physiological). PH value or chaotropic reagent) must be applied. However, if the recombinant protein is produced in a functional form, it is desirable to avoid denaturing conditions as much as possible during purification. However, three of the above examples [Hop
p et al. (1988); Martin et al. (1990); Sk.
Inner et al. (1991)] only, it has only been found that protein peptide fusions can be eluted using relatively relaxed conditions, ie competitively with eg synthetic peptides. However, a drawback still remains in these cases, because monoclonal antibodies targeting the peptide tip are either expensive or difficult to obtain in sufficient quantity.

[0005]

SUMMARY OF THE INVENTION The object of the present invention is therefore to: i) enable the binding of recombinant proteins without inhibiting their function, and ii) enable detection with readily available reagents. And iii) to develop short peptide sequences with easily controllable detection properties.

[0006]

According to the present invention, the above-mentioned problems are solved by the following amino acid sequence: Trp-X-His-Pro-
Gln-Phe-Y-Z [wherein X represents any amino acid, Y and Z both represent Gly or Y represents Glu and Z represents Arg or Lys] It Thus, within the scope of the present invention, the peptide sequence may be streptavidin, or "nuclear" -streptavidin (a product of proteolytic degradation of streptavidin) [Bayer, E .;
A. Ben-Hur, H .; Hiller, Y .; And Wilchek, M .; (1989) Bioche
m. J. 259, 369-376].

Thus, when the sequence is present in a fusion protein, this fusion protein also has a high streptavidin affinity. Corresponding fusion proteins are therefore likewise subject of the invention. In this case, the peptide sequence may be present at the carboxy terminus of the fusion protein, but theoretically may also be within the amino terminus or amino acid sequence of the protein, which has disadvantageous properties, such as biological Limited to cases that do not impair or destroy physical characteristics.

The proteins present in the fusion protein include the complete protein as well as protein mutants,
It may for example be a deletion mutant or a substitution mutant and finally it is also possible to obtain only the part of interest of the protein which is associated with the fusion protein of the invention.

The fusion protein according to the present invention can be easily detected by binding it to a complex of streptavidin and a label, and any label known to those skilled in the art can be used as the label. it can.
Other procedures for such protein detection can be performed under conditions well known to those skilled in the art.

Another subject of the invention has a DNA sequence coding for the peptide of the invention, which is expressibly arranged under the control of a suitable promoter and operator, and in the 5'-direction following this DNA sequence. , Expression vectors with several restriction cleavage positions, in particular bacterial expression vectors, which allow the introduction of other DNA sequences which code for the protein or protein part to be expressed.

The expression vector of the invention allows the DNA sequence coding for the protein of interest to be more easily placed in front of the DNA sequence coding for the peptide of the invention and may be for example Escherichia.
The fusion protein of the invention can be obtained after expression in E. coli. Insertion of the DNA sequence encoding the protein of interest at the 5'-restricted restriction site of the peptide sequence (this may be done by one of ordinary skill in the art, as would any other means of making expression vectors of the invention). A) and a fusion protein having a peptide of the present invention which provides streptavidin affinity at the carboxy terminus is obtained.

In the expression vector of the present invention, the restriction cleavage site does not necessarily have to be immediately adjacent to the first base or the last base of the DNA sequence encoding the peptide. However, preferably the restriction cleavage position is such that during transcription there is no loss of reading frame and there is a small number, preferably at most 10 additional amino acid bonds between the peptide and the amino acid sequence of the protein. Should be.

Another subject of the invention is the expression of the DNA sequence coding for the recombinant protein by a method known per se for expressing the DNA sequence coding for the fusion protein of the invention in a suitable host cell. It is a method for producing a recombinant protein. The advantage of the method of the invention is that the presence of the expression product can be easily detected by the complex of streptavidin and / or the separation to purify the desired protein as a fusion protein and streptavidin affinity chromatography. There is a point that can be done by graphy.

As mentioned above, streptavidin can be used as a complex with any label to detect the presence of the expression product, ie the fusion protein, wherein the enzyme label is within the scope of the present invention. preferable. In this case, a method known per se for detecting a protein, such as ELISA, RIA, or Western method, can also be used.

Another advantage of the method according to the invention is that the purification of the expressed fusion protein can be easily carried out by affinity chromatography on a column with immobilized streptavidin. In this case, elution can advantageously be carried out under very mild conditions, for example with the addition of biotin or a biotin-like compound or with streptavidin affinity peptides obtained by peptide synthesis.

Within the scope of the invention, elution with biotin is preferred. Also for affinity chromatography, preferably a column packed with streptavidin bound to streptavidin-agarose substrate or Eupergit ^™ C is used.

From the above description of the invention, the peptide according to the invention and the fusion protein comprising said peptide are provided with any great advantage if rapid and reliable detection of the expression product, ie the fusion protein, is possible. You can see what is going on. At this time, streptavidin can also be obtained by binding with a label such as a fluorescent label or an enzyme label. If the reagent is cheap and easy to use,
I can say. Furthermore, the expressed fusion protein exhibits an easily controllable streptavidin binding property due to the presence of the peptide according to the invention, which allows easy purification of the expression product, which is also feasible on an industrial scale. is there.

Expression of the fusion protein of the invention is facilitated by using the expression vector of the invention,
Such expression vectors are generally applicable for all proteins to be expressed. The peptides of the invention do not inhibit the biological activity of the rest of the protein part in the fusion protein and therefore do not necessarily have to be degraded before further use. However, if for any reason degradation is desired, the expression vector according to the present invention will have a further specific protease cleavage site between the restriction cleavage site for introducing the DNA sequence for the protein and the peptide coding sequence. Code D
It may also be formed to have an NA sequence. Therefore, the peptide sequence can be easily decomposed after the expression of the expression product and, in some cases, after the purification or detection of the product.

Next, the present invention will be described in detail by way of examples with reference to the drawings.

General Techniques A) Genetic Engineering Methods and Reagents DNA manipulations are carried out by conventional genetic engineering methods [Sambro
ok J. Fritisch, E .; F. And Mani
this T. (1989), Molecular C
longing: A Laboratory Manual
l. ColdSpring Harbor Labor
atory Press, ColdSpring Ha
rbor, NY]. For cloning and expression, the strain Escherichia coli K12 TG1
(SupE, hsdΔ5, thi, Δ (lac-pro
AB) [F ', traD36, proAB, lacI ^q
ZΔM15]) [Gibson, T. et al. T. (198
4) Ph. D. Theis, Cambridge U
[Neverity, England] and Escherichia coli K12 JM83 (ara, Δ (lac-pr
oAB), rpsL (= strA), φ80, LacZ
ΔM15) [Yanisch-Perron, C.I. , V
ieira, J .; And Messing, J .; (198
5), Gene 33, 103-119].
The restriction enzyme is Boehring Mannheim.
ger Mannheim, New England Biolabs (New England Biola)
bs) and Gibco BRL.
Taq DNA Polymerase is Promega
a). Restriction digestion and replication chain reaction (PC
R) was performed under the conditions recommended by the manufacturer. For site-directed mutagenesis, Kunkel
el) using the modified rule [Geisselsod
er, J.I. Witney, F .; And Yuckenbe
rg, P.I. (1987), Biotechniques.
5, 786-791]. Oligodesoxynucleotide synthesis was performed using an applied biological device / DNA automatic synthesizer. The shared streptavidin-alkaline phosphatase complex is obtained from Amersham and the streptavidin-agarose is from Biomol or Sigma. All these reagents are based on the streptavidin,
It contains a proteolytically truncated form of the protein [Bayer, E .; A. , Ben-Hur,
H. Hiller, Y .; And Wilchek, M .;
(1989) Biochem. J. 259, 369-
376].

B) Plasmid Structure The plasmid pASK46 used in this application is D1.3.
E. of the Fv fragment. An old gene structure encoding expression in E. coli [Ward E. S. , Guessow,
D. Griffith, A .; D. Jones, P .;
T. And Winter, G .; (1989) Nature
e 341, 544-546] and pASK40 [Sk
erra, A .; Pfitzinger, I .; And Pl
ueckthun, A .; (1991) Bio / Te
chnology, 9, 273-278].

The complete DNA sequence of pASK46 is shown in FIG.
It is shown in FIG. A restriction enzyme digestion map of this plasmid is shown in FIG.

Four peptides at the C-terminus of VH and two peptides at the C-terminus of VL of the D1.3Fv fragment [Boul
ot, G.I. Eisele, J .; -L. , Bentle
y, G. A. Bhat, T .; N. Ward, E .;
S. Winter, G .; And Poljak, R .; J.
(1990), J. Mol. Biol. 213, 61
7-619], six inducible plasmids of pASK46 were prepared. Structure with Peptide Tip at VH DNA on pASK46 containing base pairs 686-706
The sequence was replaced with the following sequence, each of 30 base pairs in length, encoding a nonapeptide.

[0024]

[Chemical 1]

Structure with peptide tip in VL D on pASK46 containing base pair 1127-1169
The NA sequence was replaced with the following sequences of 36 and 44 base pairs in length.

[0026]

[Chemical 2]

C) Cell Proliferation, Induction and Cytolysis For the production of recombinant proteins E. coli transformed with the corresponding expression plasmids. coli cells at 22 ° C
LB medium containing 100 μg / ml ampicillin at 50 ° C. until OD _{550 of} 0.5 was obtained. IPTG with a final concentration of 1 mM was added, the temperature was optionally lowered to 20 ° C. and induction of protein expression was continued for 3 hours. The cells were then centrifuged and resuspended in the appropriate buffer. For the preparation of the marginal cytoplasmic fraction, 1 liter of cultured cells was added to 10 ml of 50 mM Tris (pH 8.0),
Incubated on ice in 500 mM saccharose, 1 m MEDTA. The spheroplasts were sedimented by centrifugation and the supernatant was sterile filtered prior to further use. When this solution was used for purification on a streptavidin-agarose column, avidin was added to complex free biotin groups to a final concentration of 40 μg / ml. The protein solution was concentrated by ultrafiltration to a final volume of approximately 1 ml, 50 mM Tris (p
It was dialyzed against 1 liter of (H8.0) overnight. A small amount of precipitate was centrifuged [Microfuge (Microfuge, 14000 rpm, 1
0 minutes, 4 ° C.] or spin-X-filtration device (spin-X
-Filtriereinheit, Costar) was removed by filtration. To prepare a lysed portion of total cell protein, 1 liter of cultured cells was added to 50 mM
It was resuspended in Tris (pH 8.0) and ruptured at 18000 psi using a high pressure homogenizer (French press cell). Centrifuge the homogenate (45000 g,
Avidin was added to the supernatant to a concentration of 40 μg / ml for 30 minutes at 4 ° C.) for conjugation of free biotin groups. 4 ° C
After incubating for 30 minutes at 30 ° C., the protein solution was sterile filtered and applied on a streptavidin-agarose column. For SDS-PAGE of whole cell proteins,
E. 4 ml of E. coli cultured cells was added to 50 mM Tris (pH
8.0) Resuspended in 400 μl and added 100 μl × 5 SDS-PAGE application buffer (see below). Chromosomal DNA was cut by sonication before gel electrophoresis.

[0028]

【Example】

Example 1 Filter-Sandwich Test The filter-sandwich test is based on Skerra et al. [S
kerra, A .; Dreher, M .; L. And Win
ter, G.I. (1991) Anal. Biochem.
196, 151-155]. Transformation E. E. coli cells were treated with nitrocellulose-filter membrane (82 mm diameter, Schleich
er & Schuell) or applied in spots. The membrane was on LB medium agar plates containing 100 μg / ml ampicillin and 10 mg / ml glucose. The agar plates were incubated at 37 ° C for about 8 hours until small colonies were visible. In parallel with this, PB was added to the second nitrocellulose-filter membrane.
S buffer (4 mM KH ₂ PO ₄ , 16 mM Na ₂ HPO
Huner protein lysozyme (Sigma) (antigen of D1.3 antibody) 5 m in ₄ , 115 mM NaCl)
g / ml for 6 hours, then BSA in PBS buffer
(Sigma, fraction V) 3% w / v and Tween 20 (Sigma) 0.5% v / v for 2 hours. This “antibody-immobilized membrane” was washed twice with PBS, and ampicillin 100 μg / ml and 1 mM IPTG (isopropyl β-D-thiogalactopyranoside, Biomo.
liquid LB medium containing the same, and applied on an agar plate having the same medium composition. The first filter membrane with E. coli colonies covered it. Cells on this filter stack were incubated overnight (14 hours) at room temperature to express the Fv fragment with a C-terminal peptide tip. The upper filter membrane was then separated and placed on a fresh LB agar plate containing 100 μg / ml ampicillin and 10 mg / ml glucose to give E. coli
Colonies were stored at 4 ° C for later growth. The "fixed membrane" was separated from the agar and washed 3 times with PBS / Tween (PBS containing 0.1% v / v of Tween). The immobilized Fv fragment containing the peptide having streptavidin-binding activity was detected by the presence of avidin (Polyscience) 2 μg / ml which had been added in advance for 10 minutes in the presence of streptavidin-alkaline phosphatase complex (1 in PBS / Tween). : Diluted to 2000) for 1 hour. The fixed membrane was washed thoroughly (3 × PBS / Tween; 2
X PBS), then AP buffer (100 mM Tris (pH
8.8); 100mM NaCl; 5mM MgCl 2)
Incubated in 10 ml. This buffer contains BC
IP mother liquor [5-Brom-4- in dimethylformamide
Chlor-3-indolyl-phosphate-4-toluidine salt (Biomol) 50 mg / ml] 30 μl and NBT mother liquor [Nitro-blue-tetrazolium (Biomol) 75 m in 70% v / v dimethylformamide) 75 m
g / ml] 5 μl had been added [Blake, M .;
S. Johnston, K .; H. Russel-Jo
nes, G.N. J. And Gotschrich, E .; C.
(1984) Anal. Biochem. 136,
175-179]. After 30-60 minutes, the color reaction was stopped by washing several times in distilled water and the filter was air dried.

FIG. 13 shows the plasmid pASK46-p1.
41H (1), pASK46-p111H (2), pA
SK46 (3) (as a negative control) and pASK46-
E. coli transformed with p111L (4). coli TG
4 colonies of 1 are shown. Apart from the negative control,
In every other case a strong binding signal to the streptavidin complex is observed. The binding signal slightly weaker than this is due to the plasmids pASK46-p11XH, p
ASK46-p11XL and pASK46-p14XH
Was detected (not shown).

Example 2 SDS-PAGE and Western Blotting SDS-PAGE was performed in a vertical flat gel chamber with Fling,
S. P. And Gregerson, D .; S. [(198
6) Anal. Biochem. 155, 83-8
8] was used. For Western blotting, the gel after electrophoresis was immersed in a transfer buffer (electrophoresis migration buffer containing 20% (v / v) methanol). Immobilon P membrane (Millipor) for proteins by the “semi-dry” method
e company) at a constant current strength of 1 mA / cm ² at 4 ° C.
Moved in time. Next, the membrane is treated with Tween 0.5% v /
Blocked with PBS containing v and BSA 3% w / v for 1 hour at room temperature. The membrane was washed 3 times with PBS / Tween and then avidin (2 μg / ml in PBS / Tween) to complex the biotin carboxyl-carrier protein.
And incubated for 10 minutes. Next, PBS /
Incubated for 1 hour with 1: 4000 diluted streptavidin-alkaline phosphatase complex in Tween. After extensive washing (3xPBS / Tween; 2xPBS), Western blotting was performed using the BCIP / NBT protocol (see Example 1).

FIG. 14 shows the detection of the corresponding protein-peptide fusion in Western blotting. Plasmids pASK46-p141H (1), pASK46
-P111H (2), pASK46-p111L (3)
And pASK46 (4) transformed E. coli. co
The same aliquot of the peripheral cytoplasmic fraction of li TG1 cells was separated by SDS-PAGE. After transferring the protein to the Immobilon P membrane, the corresponding peptide-protein fusion was detected by the streptavidin complex. In a similar experiment, plasmid pASK46
-P11XH is pASK46-p11XL and pAS
K46-p14XH showed a slightly weaker signal. When separating whole cell proteins by SDS-PAGE,
Comparable results were obtained.

Example 3 ELISA Microtitration plate with 96 wells (Falcon # 3912)
Column 6 was loaded with 100 μl of a solution of 3 mg / ml lysozyme in 50 mM NaHCO ₃ (pH 9.6) overnight at 4 ° C. Next, skim milk powder (BioRa) in PBS at room temperature
(Company d) 2% w / v for 2 hours. Wash (3 x PBS
/ Tween), 50 μl each of the peripheral cytoplasmic cell fractions of the corresponding colonies were pipetted as a dilution series in PBS / Tween and incubated for 1 hour.
The concentration of Fv fragment in the undiluted fraction is about 50-10.
It was 0 μg / ml [evaluated by SDS-PAGE]. After washing (3 × PBS Tween), 50 μl of 1: 1000 dilution of streptavidin-alkaline phosphatase complex in PBS / Tween was added and incubated for 1 hour. Unbound complex was removed by washing twice with PBS / Tween and PBS,
NPP solution (p-nitrophenyl phosphate 0.5m
g / ml; 0.9M diethanolamine, pH 9.6;
100 μl of 1 mM MgCl ₂ ) was added with a pipette.
Generate a colored signal for 5-10 minutes, 10 mM ED
It stopped by adding 100 microliters of TA (pH8.0). The absorption value was measured using a microtitration plate photometer and the measured value was recorded as the difference value A ₄₀₅ -A ₄₅₀ after subtracting the null value for each dilution. Null value is D1.3 with no peptide tip
It was found to be constant over the entire dilution range, measured by the dilution series of the marginal cytoplasmic cell fraction containing the Fv fragment (A ₄₀₅ -A ₄₅₀ = 0.199 ± 0.00).
9).

FIG. 15 shows induced E. coli transformed with the corresponding plasmid. Figure 8 shows the observed binding signal of the streptavidin complex with the dilution series of the peripheral cytoplasmic fraction of E. coli TG1 cells.

Example 4 Protein Purification by Streptavidin Affinity Chromatography Streptavidin-agarose (about 1 / ml gel)
5 mg of streptavidin) packed column.
Equilibration was performed with 10 volumes of 0 mM Tris (pH 8.0). E. coli transformed with the corresponding plasmid. 0.5 l of the peripheral cytoplasmic cell fraction of E. coli cells was applied to the column and subsequently washed with Tris buffer. Next, the hybrid D1.3Fv fragment was treated with 1 mM iminobiotin (Sigma).
a) or 5 mM lipoic acid (Sigma) was specifically eluted with a solution of the same buffer solution. This biotin analogue binds to streptavidin much weaker than biotin itself and can therefore be regenerated by simply washing the column with 10 volumes of Tris buffer. All chromatographic steps were carried out at a temperature of 4 ° C. and a flow rate of 30 ml / h. Streptavidin affinity chromatography of the soluble portion of total cellular protein was performed under otherwise identical conditions at a flow rate of approximately 20 ml / h. The yield of purified hybrid Fv fragment is usually 0.5 mg / L OD ₅₅₀ E. E. coli culture was within range. For the streptavidin affinity purification of the D1.3Fv-lysozyme complex, D1.3
Containing the Fv (p111L) fragment and 50 mM Na ₂
H ₂ PO ₄ (pH 7.0), 115 mM NaCl, 1 m
About 3 times the molar amount of lysozyme was added to the peripheral cytoplasmic cell fraction dialyzed against M EDTA. Four
Incubate at ℃ for 1 hour, then centrifuge
The resulting protein solution was subjected to streptavidin-agarose affinity chromatography (above), using the last mentioned buffer.

FIG. 16 shows E. coli transformed with pASK46-p111H and pASK46-p111L. col
Figure 5 shows the elution profile (absorption at 280 nm) of streptavidin affinity chromatography performed on the periplasmic fraction of iTG1 cells. In this, Coomassie Brilliant Blue (Serva)
SDS-polyacrylamide gel of the fractions obtained by applying the iminobiotin solution stained with is shown. From the figure, it can be seen that the two subunits of the Fv fragment were specifically eluted together in pure form together, even though each chain of the heterodimeric protein was fused to a streptavidin affinity peptide. . p
In case of ASK46-p111L, the two chains are SDS
Had the same mobility on PAGE. Therefore, the functionality of the purified protein was also tested by ELISA (as in Example 3).
Detected by. E. coli transformed with pASK46-p111H. When the whole cell protein of E. coli TG1 cells was applied and the chromatography performed in the same manner, the recombinant protein was also obtained in pure form as a complete heterodimer.

FIG. 17 shows a similar streptavidin affinity chromatography elution profile, but in this case lysozyme, ie D1.3 Fv.
The fragment antigen was transformed into E. coli transformed with pASK46-p111L. E. coli TG1 cells were added to the marginal cytoplasmic fraction. S stained with Coomassie Brilliant Blue (Serva), illustrated by elution profile
The DS-polyacrylamide gel shows the product fraction obtained by elution with the iminobiotin solution (2), the relatively purified lysozyme (3) and the separately purified recombinant Fv fragment (4). Trace (1) represents a molecular weight standard. It can be seen that the immune complex consisting of the recombinant Fv fragment and the antigen lysozyme was specifically purified.

Example 5 Construction and use of pASK60-Strep pASK40 [Skerra, A .; , Pfitzing
er, I.I. And Pluckthun, A .; (199
1) Bio / Technology, 9, 273-2
78] starting with specific site mutagenesis and P
PASK60-Strep was prepared using CR. The restriction enzyme digestion map of pASK60-Strep is shown in FIG. 18, and the entire DNA sequence of the plasmid is shown in FIGS. The polylinker on pASK60-Strep is 3'of the region encoding the OmpA signal peptide.
It has an improved set of specific restriction cleavage sites, including two restriction positions directly at the termini, followed by a streptavidin-binding peptide "(Ser-Ala-) Trp.
-Arg-His-Pro-Gln-Phe-Gly-
Followed by the DNA sequence encoding Gly "(see Figure 30). Direct expression of the foreign gene in E. coli (ie, without the OmpA signal sequence) was determined by Xba.
It can be achieved by using the I cleavage position and reconstructing a range containing 16 base pairs between the stop codon of the lacZ minicistron and the start codon of the structural gene. In contrast, the structural gene can be cloned using the StuI or BsaI cleavage sites to create a fusion with the OmpA signal sequence encoded on pASK60-Strep. StuI (recognition sequence "AGG
CCT ") creates a blunt end after the first nucleotide of the last codon (Ala) of the signal sequence. Therefore, a precise fusion will allow compatible restriction positions (eg StuI, P).
vuII, NruI) can be created just before the structural gene. BsaI is a class IIa restriction enzyme whose cleavage site is distant from its recognition position (“GAGACC”, underlined in FIG. 30).
Cleavage with this enzyme creates a 5'-overhanging DNA end at the outermost 3'-end of the signal sequence ("GGCC", shown in lower case in Figure 30). This end does not overhang in the coding region of the mature part of the gene to be cloned. This sticky end can be ligated to the end created by the restriction enzymes EaeI and EagI. Also, for example, the DNA to be cloned
It is also possible to introduce compatible restriction positions by PCR when preparing the fragments. Such a limit position is B
saI (or other IIa class enzymes such as BspM
When the recognition sequence of I) is placed on the opposite side of the cohesive end to be made, it does not impair the mature region encoding the amino terminus of the gene. Fusion of the structural gene to the region encoding the streptavidin-binding peptide is Eco47I.
It can be achieved using the II-restriction cleavage position (recognition sequence "AGCGCT"). Thereby, claim 1
Before the actual peptide sequence described, the Ser codon ("AG
C ", shown in lower case in Figure 30) and a blunt cleavage occurs directly between the Ala codon. When reconstituting the Ser codon, various restriction positions (Eco47III) to create compatible ends are created. Besides, for example, Sca
I and NruI) can be used.

Example 6 Expression and detection of the soluble region of the LDL receptor by pASK60-Strep The human "low density lipoprotein" receptor, apart from the N-terminal signal sequence separated in the mature receptor, is:
It consists of five protein regions, the fourth of which is the transmembrane portion. For cloning of the first N-terminal protein region having 1 to 292 amino acids, the plasmid pLDLR3 (ATCC N
o57004) [Yamamoto, T .; , Davi
s, C.I. G. Brown, M .; S. , Schneid
er, W.E. J. Casey, M .; L. , Goldst
ein, J .; L. And Russell, D .; W. (1
984) Cell 39, 27-38] was used.
The corresponding DNA fragment was used as an oligodesoxynucleotide-
Primer "5'-TAG CAA CGG CCGC
AG TGG GCG ACA GAT GT "(for N-terminal: with restriction cleavage position EagI) and"5'-TTC GTT AGT ACT GCA C.
TC TTT GAT GGG TTC "(for the N-terminus: with the restriction cleavage position ScaI)
Amplified by R, isolated and the restriction enzymes BagI and S
Cut with caI. This DNA fragment was designated as pASK60-
In Strep its restriction cutting positions BsaI and Ec
o47III, E. E. coli strain K12 JM8
3 was used for cloning. After restriction analysis and DNA sequencing of the resulting plasmid, cultures are prepared from the clones and protein expression is induced as described above. The total cell protein of the obtained cells was 15% SDS-PAG.
Separated by E and transferred onto Immobilon P membrane by Western blotting. Streptavidin-alkaline phosphatase (see Example 2) could be used to detect a recombinant receptor region of approximately 40,000 daltons.

[Brief description of drawings]

FIG. 1 DNA sequence of pASK46 (showing cleavage position m)
And Figure 3 shows the amino acid sequences in three reading frames encoded by the same sequence.

FIG. 2 is a continuation of FIG.

FIG. 3 is a continuation of FIG.

FIG. 4 is a continuation of FIG.

FIG. 5 is a continuation of FIG.

FIG. 6 is a continuation of FIG.

FIG. 7 is a continuation of FIG.

FIG. 8 is a continuation of FIG.

FIG. 9 is a continuation of FIG.

FIG. 10 is a continuation of FIG.

11 is a continuation of FIG.

FIG. 12 is a view showing a restriction enzyme digestion map of pASK46.

FIG. 13 shows the detection (colony) of the protein-peptide fusion of the present invention in a filter-sandwich test.

FIG. 14 shows detection of the protein-peptide fusion of the present invention by Western blotting.

FIG. 15. Transformed induced E. binding signal of streptavidin complex with dilution of periplasmic fraction of E. coli TG1 cells (observed by ELISA)
It is a graph which shows.

FIG. 16 shows the elution profile of streptavidin affinity chromatography performed on a marginal cytoplasmic fraction similar to FIG.

FIG. 17 is a diagram showing an elution profile of the same chromatography as in FIG. 16, performed by adding an antigen (lysozyme) to the same peripheral cytoplasmic fraction as in FIG. 16.

FIG. 18 is a view showing a restriction enzyme cleavage map of pASK60-Strep.

FIG. 19: DNA sequence of pASK60-Strep (also showing the cleavage site) and 3 encoded by this sequence.
It is a figure which shows the amino acid sequence in one reading frame.

FIG. 20 is a continuation of FIG. 19;

FIG. 21 is a continuation of FIG. 20.

FIG. 22 is a continuation of FIG. 21.

FIG. 23 is a continuation of FIG. 22.

FIG. 24 is a continuation of FIG. 23.

FIG. 25 is a continuation of FIG. 24.

FIG. 26 is a continuation of FIG. 25.

FIG. 27 is a continuation of FIG. 26.

FIG. 28 is a continuation of FIG. 27.

FIG. 29 is a continuation of FIG. 28.

FIG. 30 is an explanatory diagram of the sequence of the polylinker of pASK60-Strep.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // C12N 1/21 7236-4B (C12P 21/02 C12R 1:19) (C12N 1/21 C12R 1:19)

Claims (8)

[Claims] 1. The following amino acid sequence: Trp-X-His-Pro-Gln-Phe-Y-Z [wherein X represents any amino acid, Y and Z both represent Gly, or Y represents Glu, Z is A
Representing rg or Lys]. 2. A fusion consisting of an amino acid sequence of a complete protein, a protein mutant, ie a deletion- or substitution mutant, or a protein part linked to the sequence of the peptide of claim 1. protein. 3. D encoding the peptide according to claim 1.
An NA sequence which is expressible under the control of a suitable promoter and optionally an operator and which, in the 5'- and / or 3'-direction following this DNA sequence, codes for the protein or protein part to be expressed. DNA
An expression vector having a restriction cleavage position that allows introduction of a sequence. 4. A method for producing a recombinant protein by expressing a DNA sequence encoding the recombinant protein in an appropriate host cell by a method known per se, wherein the DN encoding the fusion protein according to claim 2.
Recombinant, characterized by using the A sequence, optionally detecting the presence of the expression product by a complex of streptavidin and a label and separating the desired protein as a fusion protein by streptavidin affinity chromatography A method for producing a protein. 5. The method according to claim 4, wherein a complex consisting of streptavidin and an enzyme label is used. 6. The method according to claim 5, wherein alkaline phosphatase is used as the enzyme label. 7. Use of streptavidin-agarose substrate for affinity chromatography,
The method of claim 4. 8. Affinity chromatography elutes the fusion protein by adding the natural ligand of streptavidin, in particular biotin, its synthetic derivatives, in particular 2-iminobiotin or liponic acid; or the synthetic peptide of claim 1. The method according to claim 4.